FMR1 premutation and full mutation molecular mechanisms related to autism

Fragile X syndrome (FXS) is caused by an expanded CGG repeat (>200 repeats) in the 5′ un-translated portion of the fragile X mental retardation 1 gene (FMR1) leading to a deficiency or absence of the FMR1 protein (FMRP). FMRP is an RNA-binding protein that regulates the translation of a number of other genes that are important for synaptic development and plasticity. Furthermore, many of these genes, when mutated, have been linked to autism in the general population, which may explain the high comorbidity that exists between FXS and autism spectrum disorders (ASD). Additionally, premutation repeat expansions (55 to 200 CGG repeats) may also give rise to ASD through a different molecular mechanism that involves a direct toxic effect of FMR1 mRNA. It is believed that RNA toxicity underlies much of the premutation-related involvement, including developmental concerns like autism, as well as neurodegenerative issues with aging such as the fragile X-associated tremor ataxia syndrome (FXTAS). RNA toxicity can also lead to mitochondrial dysfunction, which is common in older premutation carriers both with and without FXTAS. Many of the problems with cellular dysregulation in both premutation and full mutation neurons also parallel the cellular abnormalities that have been documented in idiopathic autism. Research regarding dysregulation of neurotransmitter systems caused by the lack of FMRP in FXS, including metabotropic glutamate receptor 1/5 (mGluR1/5) pathway and GABA pathways, has led to new targeted treatments for FXS. Preliminary evidence suggests that these new targeted treatments will also be beneficial in non-fragile X forms of autism

Improved Methodology for Assessment of mRNA Levels in Blood of Patients with FMR1 Related Disorders

<p>Abstract</p> <p>Background</p> <p>Elevated levels of FMR1 mRNA in blood have been implicated in RNA toxicity associated with a number of clinical conditions. Due to the extensive inter-sample variation in the time lapse between the blood collection and RNA extraction in clinical practice, the resulting variation in mRNA quality significantly confounds mRNA analysis by real-time PCR.</p> <p>Methods</p> <p>Here, we developed an improved method to normalize for mRNA degradation in a sample set with large variation in rRNA quality, without sample omission. Initially, RNA samples were artificially degraded, and analyzed using capillary electrophoresis and real-time PCR standard curve method, with the aim of defining the best predictors of total RNA and mRNA degradation.</p> <p>Results</p> <p>We found that: (i) the 28S:18S ratio and RNA quality indicator (RQI) were good predictors of severe total RNA degradation, however, the greatest changes in the quantity of different mRNAs (<it>FMR1, DNMT1, GUS, B2M </it>and <it>GAPDH</it>) occurred during the early to moderate stages of degradation; (ii) chromatographic features for the <it>18S, 28S </it>and the inter-peak region were the most reliable predictors of total RNA degradation, however their use for target gene normalization was inferior to internal control genes, of which <it>GUS </it>was the most appropriate. Using <it>GUS </it>for normalization, we examined in the whole blood the relationship between the <it>FMR1 </it>mRNA and CGG expansion in a non-coding portion of this gene, in a sample set (n = 30) with the large variation in rRNA quality. By combining <it>FMR1 </it>3' and 5' mRNA analyses the confounding impact of mRNA degradation on the correlation between <it>FMR1 </it>expression and CGG size was minimized, and the biological significance increased from p = 0.046 for the 5' <it>FMR1 </it>assay, to p = 0.018 for the combined <it>FMR1 </it>3' and 5' mRNA analysis.</p> <p>Conclusion</p> <p>Our observations demonstrate that, through the use of an appropriate internal control and the direct analysis of multiple sites of target mRNA, samples that do not conform to the conventional rRNA criteria can still be utilized to obtain biologically/clinically relevant data. Although, this strategy clearly has application for improved assessment of <it>FMR1 </it>mRNA toxicity in blood, it may also have more general implications for gene expression studies in fresh and archival tissues.</p

Differential increases of specific FMR1

BACKGROUND: Over 40% of males and ~16% of female carriers of a premutation FMR1 allele (55-200 CGG repeats) will develop Fragile X associated Tremor/Ataxia Syndrome (FXTAS), an adult onset neurodegenerative disorder while, about 20% of female carriers will develop Fragile X-associated Primary Ovarian Insufficiency. Marked elevation in FMR1 mRNA transcript levels has been observed with premutation alleles, and RNA toxicity due to increased mRNA levels is the leading molecular mechanism proposed for these disorders. However, although the FMR1 gene undergoes alternative splicing, it is unknown if all or only some of the isoforms are overexpressed in premutation carriers and which isoforms may contribute to the premutation pathology. METHODS: To address this question we have applied a long-read sequencing approach using single molecule real time (SMRT) sequencing and qRT-PCR. RESULTS: Our SMRT sequencing analysis performed on peripheral blood mononuclear cells, fibroblasts and brain tissue samples derived from premutation carriers and controls revealed the existence of 16 isoforms out of 24 predicted variants. Although the relative abundance of all isoforms was significantly increased in the premutation group, as expected based on the bulk increase in mRNA levels; there was a disproportionate (4-6 fold) increase, relative to the overall increase in mRNA, in the abundance of isoforms spliced at both exons 12 and 14, specifically Iso10 and Iso10b, containing the complete exon 15 and differing only in splicing in exon 17. CONCLUSIONS: These findings suggest that RNA toxicity may arise from a relative increase of all FMR1 mRNA isoforms. Interestingly, the Iso10 and Iso10b mRNA isoforms, lacking the C-terminal functional sites for FMRP function, are the most increased in premutation carriers relative to normal, suggesting a functional relevance in the pathology of FMR1 associated disorders

Memantine Effects on Verbal Memory in Fragile X-associated Tremor/Ataxia Syndrome (FXTAS): a Double-Blind Brain Potential Study

Older FMR1 premutation carriers may develop fragile X-associated tremor/ataxia syndrome (FXTAS), a neurodegenerative disorder manifesting cognitive deficits that often subsequently progress to dementia. To date, there is no specific treatment available for FXTAS. Studies have demonstrated the premutation-associated overactivation of glutamatergic receptors in neurons. Memantine, a NMDA receptor antagonist approved for treatment of Alzheimer's disease, thus was tested in the first placebo-controlled, double-blind, randomized clinical trial in FXTAS. Prior event-related brain potential (ERP) studies in FXTAS found reduced N400 repetition effect, a glutamate-related electrophysiological marker of semantic priming, and verbal memory processes. This substudy of the randomized clinical trial of memantine in FXTAS sought to use the N400 repetition effect to evaluate effects of chronic memantine treatment on verbal memory. Subsequent recall and recognition memory tests for the experimental stimuli were administered to characterize verbal memory. Data from 41 patients who completed the 1-year memantine trial (21 on memantine) and also completed longitudinal ERP studies were analyzed. Results showed treatment-associated benefits on both cued-recall memory and N400 repetition effect amplitude. Importantly, improvement in cued recall was positively correlated with amplitude increase of the N400 repetition effect. The placebo group, in contrast, displayed a significant reduction of the N400 repetition effect after 1 year. These results suggest that memantine treatment may have beneficial effects on verbal memory in FXTAS. Additional studies of memantine, perhaps in combination with other therapeutic agents, appear warranted, as symptomatic treatments and neuroprotective treatments are both needed for this recently recognized neurodegenerative disorder